PROJECT SUMMARY/ABSTRACT This MERIT extension remains focused on the development and application of novel fluorescence methods to measure solute mobility and interactions in live cells and tissues. The aims of the research are a direct extension of ongoing work to understand the determinants of extracellular space (ECS) diffusion and volume (Aim 1), plasma membrane protein diffusion (Aim 2) and protein-protein interactions (Aim 3). In Aim 1, novel optical methods will be applied to study regulated ECS diffusion, volume and ionic homeostasis in brain and tumor. We will apply microfiberoptic methods, polarization correlation microscopy, and K'^-sensing fluorescent indicators to study ECS regulation in brain and tumor, addressing questions regarding the microviscosity of the extracellular matrix, the dynamic changes in ECS volume and [K*] during neural signal transduction, and the influence of cellular crowding on macromolecule diffusion in the ECS. In Aim 2, single-molecule fiuorescence methods will be applied to investigate the determinants of membrane protein translational and rotational diffusion. Building on quantum dot-single particle tracking (SPT) studies of aquaporin-4 (AQP4) water channels, we will use multi-color SPT and quantum rod polarization correlation microscopy to investigate the determinants of AQP4 translational and rotation diffusion in live cells, and to study AQP diffusion in lamellipodia of migrating cells and the determinants of AQP4 supramolecular assembly. In Aim 3, single-molecule fluorescence methods will be applied to investigate protein-protein interactions of membrane water and ion transporters. We have implemented methodology to quantify protein-protein interactions in live cells, including multicolor SPT, two-color FCS, and super-resolution microscopy. Building on our recent studies of AQP water channels and CFTR Cl channels, we will investigate the size and dynamics of AQP4 supramolecular assembly, and the cytoskeletal and other interacting proteins of AQP4 and CFTR. These data will provide molecular-level information about the mechanisms and biological consequences of AQP4 and CFTR protein-protein interactions. In addition, the proposed studies will establish novel and widely applicable methodology to measure protein dynamics and interactions in cell plasma membrane and in the extracellular space.